The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure–activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs – including BPP-10c – are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively.The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril.The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle. Spermatogenesis takes place in the seminiferous epithelium of the mammalian testis. The germ cells residing in the basal compartment must traverse the blood-testis barrier (BTB) and enter the adluminal compartment for further development into round, elongated spermatids . The intTestis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in maturing germ cells and spermatozoa, but not in Sertoli cells, Leydig cells or any other somatic cell in male adults, suggesting that it is related to the spermiogenesis process -7. ExperSomatic angiotensin I-converting enzyme (sACE) is a well-characterized zinc dipeptidyl carboxypeptidase that plays a pivotal role in the regulation of blood pressure by converting angiotensin I into angiotensin II and by inactivating bradykinin . sACE haBothrops jararaca snakes were the first natural sACE inhibitors described. Studies of their structure–activity relationships were the basis for the development of antihypertensive drugs, such as captopril [Bradykinin potentiating peptides (BPPs) from aptopril . Typicalaptopril .i(app) = 0.5 nM) [We found that BPP-10c (<ENWPHPQIPP) is able to distinguish between the two domains of sACE and displays distinct hypotensive effects on rats ,20. In a 0.5 nM) . Captopr 0.5 nM) . In rece 0.5 nM) -25. Thesin vitro nanomolar range inhibition of human tACE by BPP-5a (<EKWAP) and BPP-9a (<EWPRPQIPP) has been reported, there are no reports on the possible effects of BPPs in the structure and function of the seminiferous epithelium [Considering the structural similarity between the C-domain of sACE and tACE, it was observed that tACE male knockout mice were severely hypofertile, tACE was exclusively expressed in maturing germ cells, BPP-10c had selectivity for the active site at the C-domain of sACE and it could be internalized by different cells, and ACE inhibitors could affect the function of the seminiferous epithelium, particularly spermiogenesis ,22,24,25ithelium . Thus, tad libitum, and were subjected to a light–dark cycle (12 hours each). The experimental protocols were performed in accordance with the guidelines of the Butantan Institute for the humane use of laboratory animals and were approved by local authorities (protocol number 369/07).Male Swiss mice (weighting 30 to 35 g) were bred at the Butantan Institute . Animals were housed at a temperature of 22°C, had access to water and food All chemicals were of analytical reagent grade, purchased from Calbiochem-Novabiochem Corp. (USA), Merck (USA) and Sigma–Aldrich Corp. (USA) for peptide synthesis; captopril and bradykinin were purchased from Sigma Chemical Co (USA).2O (1:1000) and (B) TFA/ACN/H2O (1:900:100). The column was eluted at a flow rate of 5 mL/minute over 20 minutes with a 10 to 50% gradient of solvent B, and the effluent was detected with an SPD-10AV Shimadzu UV–vis detector, monitored by absorbance at 220 nm. The molecular weight and purity of synthetic peptide were checked via MALDI-TOF mass spectrometry using an Ettan MALDI-TOF/Pro system  and cinnamic acid as a matrix. The peptide concentration was determined by amino acid analysis after acid hydrolysis in vacuum-sealed tubes at 110°C for 22 hours with HCl 6 N containing 1% phenol. Samples were subjected to amino acid analysis using a pico Tag station.BPP-10c (<ENWPHPQIPP) was synthesized using automated solid-phase synthesis via Fmoc (9-fluorenylmethyloxycarbonyl) strategy . The finet al. [2 asphyxiation after treatment and testes were collected for morphological, morphometric and immunohistochemical analyses of seminiferous epithelium. BTB permeability studies were carried out in mice treated with BPP-10c, captopril , lipopolysaccharide  or vehicle (0.91% w/v aqueous sodium chloride solution – negative control) for 15 days. All treatments and experiments were performed in duplicate or triplicate.Male adult mice (30–35 g) were assigned to groups  and treated for 15 days (once a day) by intraperitoneal injection with 4.7 μmol/kg/day of BPP-10c or captopril, diluted in 100 μL of 0.91% w/v aqueous sodium chloride solution. The control group consisted of treatment with vehicle only. The dose of BPP-10c used in the experiments was in agreement with Silva et al. . Mice weThe testes of mice treated with BPP-10c, captopril or vehicle were immediately immersed and fixed in Bouin’s solution for 24 hours. The samples were dehydrated in ethanol, and embedded in Paraplast®  and sectioned at 4 μm thickness. Histological sections were stained with periodic acid-Schiff’s (PAS) with Harris hematoxylin counterstaining , or Mallory's trichrome stain . Images were taken using a Pixera camera  mounted on a Zeiss Axioskop 2 photomicroscope and captured with a Intel Pentium® computer using Adobe Photoshop 7.0.1 .http://rsb.info.nih.gov/nih-image). The germ cell nuclei  and Sertoli cell nucleoli present at stages I, V, VII/VIII, and XII of the seminiferous epithelium cycle were counted using Adobe Photoshop 7.0.1. Total support capacity of each Sertoli cell was obtained by the ratios of total number of germ cells to total number Sertoli cell nucleoli for each stage.The stages of the seminiferous epithelium cycle were characterized based on the development of the acrosomic system and morphology of the developing spermatid nucleus . Four sp2O2 for 30 minutes, at room temperature, to block endogenous peroxidase activity. Samples were then washed in phosphate-buffered saline pH 7.4 (PBS), two times for 5 minutes each, and immersed in a solution containing 5% fat-free dry milk  in PBS for 15 minutes to block non-specific binding sites.Testis sections from mice treated with BPP-10c, captopril or vehicle were processed according to the Streptavidin-Biotin-peroxidase Complex (SBC) protocol. After deparaffinization and dehydration, the sections were pretreated with 0.03% HSections were incubated overnight at 4°C with anti-rabbit claudin-1 antiserum  diluted (1:250) in 0.05 M Tris–HCl with 1% bovine serum albumin (BSA). They were washed in PBS three times for 5 minutes and incubated with the diluted biotinylated anti-rabbit IgG for 30 minutes, then washed in PBS three times for 5 minutes and incubated for 30 minutes with Streptavidin-biotin-peroxidase complex. Immunoreactive sites were revealed using a buffered solution of 3,3’-diaminobenzidine–tetrahydrochloride (DAB) . The sections were dehydrated, mounted and analyzed in a Zeiss Axioskop 2 photomicroscope and the images were captured by Pixera . As negative control, normal rabbit IgG (Vector Laboratories) was used instead of the first antibody in every experiment. Hematoxylin was used for counterstaining. We also performed immunoblot analysis of mouse testis lysate to assess the specificity of anti-rabbit claudin-1 antibody.Mice treated with BPP-10c, captopril, LPS or vehicle were perfused with a hypertonic fixative of 5% glutaraldehyde, 0.05 M sodium cacodylate, and 10% dextrose . Testes t-test . The criteria for statistical significance were set at p < 0.05.All statistical evaluations were performed either by one-way analysis of variance (ANOVA) followed by the Bonferroni range test or by Student’s The seminiferous tubules observed in animals treated with vehicle Figure  – A and Epithelium height increased and lumen diameter decreased in the testes of the animals treated with BPP-10c, but no alteration was detected in the tubule diameter when compared with vehicle Figure  – A. FurClaudin-1 was observed in the mouse testis tissue lysates through immunoblot analysis using anti-claudin-1 antibody, detected as a single band at approximately 20 kDa Figure –A. The lThe barrier can be visualized in testes fixed with a dextrose-containing hypertonic fixative. The permeability of BTB was examined in testicular sections of mice treated with vehicle, captopril, BPP-10c or LPS. Cells directly exposed to the fixative, such as those on the basal side of the barrier, suffer the osmotic effects of the dextrose and shrink, leaving large intercellular gaps. Because dextrose cannot transverse the BTB, cells on the luminal side are protected from the hypertonicity of the fixative and maintain close apposition with one another. The BTB was maintained in testes of mice treated with vehicle, captopril and BPP-10c .The authors declare that there are no competing interests.JMG carried out most experiments assisted by other researchers. FCVP, MIB and CL contributed to the design of experiments. ACMC established the conditions for the study. CAS established the conditions for the study, was responsible for drafting the manuscripts, reading it and for the editorial corrections. All authors read and approved the final manuscript.


Heroin use among young women of reproductive age has drawn much attention around the world. Although methadone is widely used in maintenance therapy for heroin/morphine addiction, the long-term effects of prenatal exposure to methadone and preventative therapy remain unclear. For revealing this question, female pregnant Sprague–Dawley rats were sub-grouped to receive (1) vehicle, (2) methadone 5 mg/kg at embryonic day 3 (E3) and then 7 mg/kg from E4 to E20, (3) dextromethorphan (DM) 3 mg/kg, and (4) methadone + DM (the rats received methadone followed by DM treatment), subcutaneously, twice a day from E3 to E20. The body weight, natural withdrawal, pain sensitivity, ED50, conditioned place preference and water maze were conducted at different postnatal stages (P1 to P79) of offspring. The quantitative real-time RT-PCR and electrophysiology were also used to measure the gene expression of opioid receptors in the spinal cord and changes of LTP/LTD in the hippocampus, separately.Prenatal exposure to methadone or DM did not affect survival rate, body weight, water maze and LTP or LTD of offspring. However, prenatal methadone significantly increased the withdrawal symptoms, pain sensitivity, addiction liability and decreased the mRNA expression of pain related opioid receptors. Co-administration of DM with methadone in the maternal rats effectively prevented these abnormalities of offspring induced by methadone.Our study clearly showed that co-administration of dextromethorphan with methadone in the maternal rats prevented the adverse effects induced by prenatal methadone exposure. It implies that dextromethorphan may have a potential to be used in combination with methadone for maintenance treatment in pregnant heroin-addicted women to prevent the adverse effects induced by methadone on offspring.The online version of this article (doi:10.1186/s12929-015-0126-2) contains supplementary material, which is available to authorized users. N-methyl-D-aspartate (NMDA) receptor, with the majority of the μ-opioid receptor agonist activity residing in the R-isomer [Abuse of opioids causes serious social and health problems around the world. Methadone is a synthetic mu- (μ-) opioid receptor agonist that has been used for maintenance therapy in heroin addicts (including pregnant women) for more than 40 years . AlthougR-isomer . MethadoR-isomer ,10. SomeR-isomer -13. AddiR-isomer . The dosR-isomer , some caR-isomer ,17. SeveR-isomer -20. ThisDextromethorphan (DM), a commonly used antitussive agent, is without opioid-like activity . Both DMAlthough methadone is also a μ-opioid agonist like morphine, the effects of DM in prenatally methadone-exposed offspring have not been explored. Therefore, in the present study, we aimed to investigate if the prenatal co-administration of DM with methadone prevents or decreases the possible adverse effects induced by methadone on the offspring, such as withdrawal symptoms, changes of pain sensitivity, tolerance, addiction liability (reward), memory impairment, etc.ad libitum. The care of animals was carried out in accordance with institutional and international standards ; the animal protocol was approved by the IACUC of the National Health Research Institutes (NHRI-IACUC-099087-A). All experiments were performed following the schedule below.Pregnant Sprague–Dawley rats  and their offspring were used in this study. All animals were housed in an animal room with a 12-h light/dark cycle (light on 07:00–19:00), a temperature of 25°C, and humidity of 50 ± 10%. Pregnant rats were kept individually in separate cages, and their male offspring were housed 2–3 per cage after weaning. A diet  and water were provided s.c.) in a volume of 1.0 ml/kg of body weight.Methadone HCl  or dextromethorphan HBr  were dissolved in sterilized distilled water and were administered subcutaneously (s.c. injected with either vehicle or drug(s) during the gestational period (E3–E20). Vehicle control group rats received distilled water, 1 ml/kg (s.c.), twice a day. Methadone-group rats received 5 mg/kg at E3 and then 7 mg/kg from E4 to E20 (s.c), twice a day. DM group rats received 3 mg/kg of DM (s.c.), twice a day. In the methadone + DM group, the rats received methadone followed by DM treatment twice a day. The offspring were weaned at day 28 after birth and housed 2–3 per cage after weaning until use. The offspring from the same dam were randomly assigned to different experiments to avoid the litter effect .Pregnant Sprague–Dawley rats, weighting 200–250 g (10–12 weeks old), were assigned to four groups randomly. Each group was Natural withdrawal behaviors of the offspring were observed for 15 min within 24 h after birth. One pup was chosen randomly and removed from the dam before being placed on a white filter paper. Head moves, moving paws, rolling, stretching, twisting, and walking behaviors were observed and counted for 15 min. There were at least 11 pups in each group.Tail-flick test: A light beam was focused on an animal’s tail until that the rat flicked its tail. The recorded time was a measure of the pain threshold. The tail-flick apparatus  was used in the study. The basal latency of most rats was around 3–3.5 sec; a cutoff time of 10 sec was used to avoid tissue damage.Hot-plate test: In this study, a hot-plate apparatus  was used. The animal was placed on the hot plate and the time was measured until the animal started to shake or lick its hind paw or jump from the hot plate. To avoid tissue damage, the animal was removed immediately from the metal surface after it showed pain-related behavior or reached the cutoff time (30 sec).50 value was used as an index of the antinociceptive effect of methadone and this was determined by the up-down method [50 after sub-chronic treatment of methadone  (day 79) and ED50 before CPP experiment and sub-chronic methadone treatment (day 61).The EDn method  with thes.c.) in the afternoon and put in another chamber of the CPP box for 40 min everyday for 6 days. CPP tests were performed and recorded for 15 min on the day before and the day after conditioning for 6 days.In this study, we used the CPP test to evaluate methadone-induced reward in the adult male offspring p64-p71). Details are shown in the Additional file . DetailsThe water maze was circular, had a diameter of 180 cm and a depth of 60 cm, and was filled with water (21–23°C) to a depth of 40 cm. Spatial cues were located on the walls. The total pool was divided into 4 quadrants , which were designated as zones 1–4, respectively. The transparent platform (2 cm below the surface of the water) was placed in the zone 1 area (NE quadrant). Rats received 5 days of training for two trials per day. The time for the animal to find the platform was recorded, and the maximal duration for each trial was 60 sec; then the animal was put on the platform for 10 sec to allow the animal to look around. On day 6, the platform was removed and the time the animal spent in zone 1, the previous location of the platform, was determined. All parameters were automatically recorded and analyzed by video tracking software .The details of the electrophysiology recording protocol are described in the Additional file TM cDNA Synthesis Kit . The SYBR green system  was used to determine the Ct (threshold) value. Primer sequences for NOP receptor (Oprl1) , μ-opioid receptor (Oprm1) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (housekeeping gene) are presented in Table Gapdh. ΔΔCt = ΔCt (target genetreat- GAPDHtreat)-ΔCt (Controlcontrol-GAPDHcontrol). The fold change was measured as 2−ΔΔCt.Details of qPCR protocol are described in the Additional file ptor Oprl  = 3.19; p = 0.08). The litter sizes in all tested groups were not significantly different in current study. Similarly, the body weights of all tested groups at p14  = 0.47; p = 0.71), p30  = 2.30; p = 0.09), and p60  = 0.006; p = 0.999) showed no significant difference  = 5.00; p < 0.05) or p60  = 2.85; p < 0.05). Prenatal exposure to DM did not alter the pain threshold of offspring significantly. However, prenatal exposure of DM in conjunction with methadone from E3-E20, prevented the changes induced by prenatal methadone exposure alone either partially or completely in the tail flick and hot plate tests, respectively.Prenatal methadone-exposure significantly decreased the pain threshold of offspring determined by either tail-flick Figure A or hot- = 56.35; p < 0.001) and NOPR  = 60.53; p < 0.001)  and nociceptin/orphanin FQ receptors (NOPR) in the spinal cord of male offspring. Our results showed that prenatal exposure to methadone caused a significant decrease in the gene expression (mRNA level) of MOR  = 6.59; p < 0.001)  = 11.89; p < 0.001).The CPP test was used to determine the rewarding effect of methadone in the offspring. We found that when methadone  was given once daily and conditioned for 6 days, it significantly increased the preference time for the drug-paired compartment in all groups tested (Fi.e. increased the pain sensitivity). In this study, the methadone-induced tolerance was measured in offspring of all groups. As shown on Table 50 of all the groups before the CPP experiment were similar (1.58–1.77 mg/kg). After the CPP experiment and sub-chronic treatment of methadone, the ED50 of all the groups increased 1.34–1.84-fold, as shown on Table 50 after sub-chronic methadone administration.According to the results shown in Figure  = 0.66; p = 0.9226) and there was also no significant difference for time spent in the platform quadrant (without platform) on day 6  = 0.89; p = 0.45) (Figure A water maze was utilized to test the spatial learning and memory in prenatal methadone-exposed offsprings. We found no difference in the water maze test among all groups tested Figure A and B. 3,32 = 0.66; The goal of this study is to evaluate the effects of co-administration of DM with methadone in the prenatal stage on offspring ,29,31. TThe survival rate and body weights of offspring in methadone group were not different from those of the vehicle control group. Prenatal exposure to DM or methadone + DM at the doses we used did not change the survival rate or body weight of offspring Figure . FurtherThe changes in pain sensitivity in prenatal methadone-exposed animals might be because methadone changes the expression of opioid receptors. MOR and NOPR are expressed early in the brain and spinal cord at the embryonic stage ,33. ThesOn the other hand, although our results showed that the mRNA of NOPR and MOR were also reduced in the spinal cord of prenatal DM-exposed rats, the pain sensitivity did not show significant change in this group of animals. These results imply a complicated cross-talk of cellular signaling to behavioral expression and need further exploration. In a brief summary, the increase of pain sensitivity in the prenatal methadone-exposed animals may be partly due to the reduction of both NOPR- and MOR-related signals.Besides pain regulation, prenatal opioid-exposed offspring might also change their reward sensitivity to opioids, such as offspring from morphine-addicted mother becoming more sensitive to morphine-induced reward ,49. In tSeveral studies have found that the NAS of offspring was related to the dose of methadone used in the mother ,19,50. ARecently, the relationship between NMDA receptors and opioid systems has been studied. Morphine tolerance could be attenuated by resveratrol via inhibiting neuroinflammation and down-regulating the expression of NR1 and NR2B subunit of NMDA receptors . MorphinCompared with our previous prenatal morphine-exposed studies ,31, methIn summary, this study showed that co-administration of DM with methadone in the maternal rats significantly decreased the effects of prenatal methadone on pain sensitivity, natural withdrawal, and reward in the offspring. In conclusion, this study reveals that DM may have great potential to be used in combination with methadone maintenance therapy in pregnant heroin abusers to reduce the side effects of methadone on the offspring.


Falls are the leading cause of unintentional injuries in the U.S.; however, national estimates for all community-dwelling adults are lacking. This study estimated the national incidence of falls and fall-related injuries among community-dwelling U.S. adults by age and gender and the trends in fall-related injuries across the adult life span.falls, and pooled data from the NHIS was used to calculate estimates of fall-related injuries in the U.S. and related trends from 2004–2013. Costs of unintentional fall-related injuries were extracted from the CDC’s Web-based Injury Statistics Query and Reporting System.Nationally representative data from the National Health Interview Survey (NHIS) 2008 Balance and Dizziness supplement was used to develop national estimates of falls at a rate of 37.2 falls per 100 person-years. On average, 9.9 million fall-related injuries occurred each year with a rate of 4.38 fall-related injuries per 100 person-years. In the previous three months, 2.0% of older adults (65+), 1.1% of middle-aged adults (45–64) and 0.7% of young adults (18–44) reported a fall-related injury. Of all fall-related injuries among community-dwelling adults, 32.3% occurred among older adults, 35.3% among middle-aged adults and 32.3% among younger adults. The age-adjusted rate of fall-related injuries increased 4% per year among older women (95% CI 1%–7%) from 2004 to 2013. Among U.S. adults, the total lifetime cost of annual unintentional fall-related injuries that resulted in a fatality, hospitalization or treatment in an emergency department was 111 billion U.S. dollars in 2010.Twelve percent of community-dwelling U.S. adults reported falling in the previous year for a total estimate of 80 million Falls and fall-related injuries represent a significant health and safety problem for adults of all ages. The findings suggest that adult fall prevention efforts should consider the entire adult lifespan to ensure a greater public health benefit. Several research studies and prevention programs have focused on fall prevention and examined interest. However,interest.–14 Howevfall-related injuries by age and gender. Using the National Health Interview Survey (NHIS), we estimated the national incidence of falls, the incidence of injuries resulting from falls, injury types, and the trend over time by age and gender among community-dwelling U.S. adults. As another measure of burden, we also report the national cost burden of fall-related injuries by age-group using the Centers for Disease Control (CDC) and Prevention’s Web-based Injury Statistics Query and Reporting System (WISQARS).In addition, despite being the leading cause of injury-related emergency department visits in nearly every adult age stratum, there remains a lack of detailed national estimates of The NHIS is an in-person household survey which is the principal source of information on the health of the civilian, noninstitutionalized population of the United States. For eachIn 2008, with sponsorship by the National Institute on Deafness and Other Communication Disorders (NIDCD), the SA Core of the NHIS included questions relating to balance/fall issues in adults in the previous 12 months. We defined Fallers  and Non-Fallers and the number of falls in the previous 12 months, regardless of injury, using the 2008 Balance and Dizziness supplement. Fallers and number of falls were determined by following questions:“During the past 12 months, have you fallen at least once a month on average?”“During the past 12 months, how many times have you fallen?”During the past 12 months, have you fallen at least once a month on average?) were designated Fallers and their number of falls in the past 12 months was set to 12, and they were not asked the second question.SA who responded “yes” to the first question . SA responding “0” to the second question were designated as Non-Fallers; and SA responding “1 time,” “2 times,” “3–4 times,” “5–7 times,” and “8 or more times” to the second question were designated as Fallers and the number of falls in the past 12 months were quantified as 1, 2, 3.5, 6, and 10, respectively, to calculate the total number of falls and the rate of falls per 100 persons.SA who responded “no” to the first question were then asked the second question . Persons experiencing a fall-related injury episode and the number of fall-related injury episodes between 2004 and 2013 were identified using ICD-9-CM codes: E880 -E888, “Accidental Falls” and ICD-9-CM codes 800–999, “Injury and Poisoning” were used to identify injury types. All analyses were performed using the Sample Adult subset to avoid proxy responses.Over the study period (2004–2013), out of 289,187 sampled adults in the NHIS, 3,408 reported a fall-related injury in the previous three months. The average response rate was 66% during this period.http://www.cdc.gov/injury/wisqars/cost_help/calculations_toc.html. The total lifetime costs of unintentional fall-related injuries that resulted in a fatality, hospitalization or treatment in an emergency department in each age group were divided by the total population in the age group to calculate lifetime costs of fall-related injuries per person.Costs of unintentional fatal and non-fatal fall-related injuries were extracted for the year 2010 from the CDC’s Web-based Injury Statistics Query and Reporting System (WISQARS). The estifall in the past 12 months, 2) the number of falls experienced by adults in the past 12 months and the rate of falls per 100 person-years, 3), the number and proportion of adults 18+ reporting a fall-related injury in previous 3 months, and 4) the number of fall-related injuries to adults 18+ in the previous three months and the rate of fall-related injuries per 100 person-years. We annualized the estimates obtained from data pooled over multiple years (2004–2013). We examined the trend in the rate of fall-related injuries from 2004 to 2013 for 6 age-gender groups . Poisson generalized linear models which accounted for sampling weight and clustering were used to examine the association between survey year and the rate of fall-related injuries in each age-gender group. The models were adjusted for age to account for change in average age by survey year in each age-gender group. We also estimated the number and proportion of injury types in these six age-gender groups.Using sample weights available in the NHIS data and the PROC SURVEYMEANS procedure in SAS 9.4, we estimated: 1) the number and proportion of adults (18+ years of age) who reported a Based upon the data from the NHIS 2008 supplement, 11.9% of community-dwelling U.S. adults fell in the previous 12 months . The proThe proportion of male and female Fallers generally increased with each decade of age range after the 25–34 year age-group . SimilarBased upon this pooled NHIS annual survey data (2004 to 2013), 1.04% of community-dwelling U.S. adults reported a fall-related injury in the previous three months, resulting in approximately 9.9 million fall-related injuries annually . An estiThe proportion of persons injured due to a fall increased monotonically with age after the 25–34 year age-group . In the In the overall adult population, the most common fall-related injury was “Sprains And Strains Of Joints And Adjacent Muscles” 38%, . HoweverAmong U.S. adults, the total lifetime cost of annual unintentional fall-related injuries that resulted in a fatality, hospitalization or treatment in emergency department was 111 billion U.S. dollars . The higfalls at a rate of 37.2 falls per 100 person-years. While this is the best estimate available at the national level and demonstrates a significant exposure to falling, it is likely an underestimate of the number of individuals every year exposed to falls. Fall incidents, especially those without an injury, are difficult to recall over a 12-month period. The proportion of Fallers in the oldest age group (65+) was 16.4%. This estimate, for the aforementioned reasons, is comparably lower than other estimates for this age group; a number of prior studies have reported that about a third of community-dwelling adults over 65 years of age report falling in one year.[Using a special supplement of the NHIS, one of the largest national health surveys in the U.S., we found that 12% of community-dwelling U.S. adults reported falling in the previous year for a total estimate of 80 million one year.We found that the proportion of Fallers in the previous 12 months was 10.6% in the 18–44 year age-group, and 11.4% in the 45–64 year age-group. For comparison, a study using the data from the Baltimore Longitudinal Study on Aging found that 18.5% of young adults (20–45 years), 21% of middle-aged adults (46–65 years) and 35% of older adults (65+) reported one or more falls in the previous two years. Some of Regardless of the variability in the findings across studies and the values we estimated from the NHIS 2008 Balance and Dizziness Supplement, the number of community-dwelling adults who report falling is high and represents a significant public health concern for not only older adults but for adults of all ages. As the number and the rate of fall-related injuries have increased in recent years, it is possible that the number of falls and falls rates have also increased from 2008. Therefore, in addition to collecting surveillance information about fall-related injuries, it is essential to also regularly collect information on falls as the high frequency of falls makes it possible for national and state-based surveillance systems to provide stable estimates, even for smaller geographic regions and, thus, assess risk factors for falls and trends over time. In addition, an emphasis on falls also provides the opportunity to efficiently conduct prospective studies and randomized controlled trials to evaluate fall prevention strategies in older adults as well as among middle-aged and younger adults. Finally, regular assessment of falls across all ages may help in early interventions to reduce the risk of falls and, ultimately, prevent fall-related injuries.fall-related injuries were reported each year among community-dwelling U.S. adults at a rate of 4.38 fall-related injuries per 100 person-years, according to the pooled NHIS data from 2004 to 2013. Corso et al. also reported a similar rate of fall-related injuries in the U.S. (4.2 per 100 person-years) using data from the Medical Expenditure Panel Survey, Healthcare Cost and Utilization Project—Nationwide Inpatient Sample and National Electronic Injury Surveillance System—All Injury Program.[About 9.9 million  Program. The Cent Program.In the previous three months, 2.0% of older adults, 1.1% of middle-aged adult and 0.7% of young adults reported a fall-related injury and the rate of fall-related injuries increased with age. In general, older adults are more vulnerable to falls and injuries resulting from them. The injuries from falls are also more severe in older adults. Therefore, the focus of research and fall prevention has been predominantly on the older adult population with onlMiddle-aged adults may progressively start to experience higher incidence of disease and medication use along with decreasing postural stability, proprioception, and balance.–37 FurthThe cost of injury data from WISQARS showed that the cost of fall-related injuries per person among middle-aged adults was about half of the cost among older adults. A recentWe observed an increasing trend (age- and gender-adjusted) in the rate of fall-related injuries from 2004 to 2013 among community-dwelling U.S adults  and, particularly, among older adults. We observed a similar magnitude of increasing trend among older adults of both genders (4% per year for older women and 5% per year for older men). However, the trend was not statistically significant for older men at 0.05 level. There was a smaller estimated number of men in comparison to women among those in the 65+ year age group ,” “5–7 times ,” and “8 or more times ” to assign the number of falls. For more than 12 falls in the previous year, the number of falls was set to 12. The assignment of the number of falls based on stratum may have led to overestimation or underestimation of the total number of falls. We conducted a sensitivity analysis using the lower and upper boundaries of each stratum to assign the number of falls and observed that changing the assignment protocol did not significantly affect the overall estimate of the number of falls.The NHIS does not include institutionalized individuals. Elderly individuals who fall and get injured are more likely to be institutionalized , 60 and,This study provides national estimates of falls and fall-related injuries among community-dwelling U.S. adults across the adult life span. Based on the 2008 NHIS Balance and Dizziness Supplement and the pooled NHIS annual data, about 12% of community-dwelling U.S. adults fall every year, experiencing an approximate national total of 80 million falls, and 9.9 million fall-related injuries occur every year among community-dwelling U.S. adults at a rate of 4.38 injuries per 100 person-years. In the previous three months, 2.0% of older adults, 1.1% of middle-aged adult and 0.7% of young adults reported a fall-related injury. Of all fall-related injuries to adults, 32.3% occurred among older adults, 35.3% among middle-aged adults and 32.3% among younger adults. Age-gender adjusted rates of fall-related injuries are increasing in the U.S.; more research is needed to understand possible reasons and to develop and implement effective fall and fall-related injury prevention strategies across all ages to significantly reduce the burden of injuries from falls.


Background. Evidence is accumulating in favour of a link between erectile dysfunction (ED) and coronary artery diseases. We investigated the presence of cardiac injury in patients who have had arteriogenic and nonarteriogenic ED using the hs-Tn levels. Methods. The diagnosis of ED was based on the International Index of Erectile Function 5-questionnaire (IIF-5) and patients were classified as arteriogenic , nonarteriogenic , and borderline  patients in relation to the results of echo-color-Doppler examination of cavernous arteries. The level of hs-TnT and hs-TnI was measured in 120 men with a history of ED of less than one year with no clinical evidence of cardiac ischemic disease. Results. The levels of both hs-TnT and hs-TnI were within the reference range and there was no significant (P > 0.05) difference between patients of the three groups. The hs-CRP values were higher in A-ED men compared with NA-ED (P = 0.048) but not compared with BL-ED (P = 0.136) and negatively correlated with IIF-5 . Conclusions. In ED patients of the three groups the measurement of hs-Tn allows us to exclude the presence of cardiac involvement at least when the history of ED is less than one year and the men are without atherosclerotic risk factors. Erectile dysfunction (ED) is now considered an early manifestation of a largely subclinical systemic vascular disorder affecting also the penile arteries. Indeed, ED shares with other vascular diseases, mainly coronary artery disease (CAD), common risk factors –4 and a Cardiac troponin T (TnT) and troponin I (TnI) are the markers recommended for diagnosis of acute myocardial infarction . With thOut of a series of 590 filed ED cases studied in the period of October 2009 to June 2011, we included in this study all patients with a history of ED of more than 3 months but less than one year. Exclusion criteria were clinical evidence from the patient's clinical history of coronary artery disease, dyslipidemia, arterial hypertension, malignancies, renal failure, congestive heart failure, systemic inflammatory disease, rheumatic disease, infection diseases, anemia, chronic liver diseases, arrhythmias, trauma, and no current smoking and vitamins supplementation or chronic drug assumption –19. Thisβ-estradiol, urinalysis and 24 h urinary albumin excretion , the International Index of Erectile Function questionnaire (IIEF), and echo-color-Doppler of both cavernous arteries.On the basis of the given inclusion and exclusion criteria reported above, we selected a total of 120 ED patients who were included in the study. Their clinical characteristics are summarized in μg prostaglandin E1 (PgE1), and the peak systolic velocity (PSV) and end-diastolic velocity (EDV) were recorded 5, 10, 15, 20, and 25 min after the injection in the proximal portion of the penis. Patients were classified as “nonarteriogenic” (NA-ED) when their PSV was ≥35 cm/sec or ≤35 cm/sec but >25 cm/sec with concomitant EDV ≤0 cm/sec, “arteriogenic” (A-ED) when their PSV was ≤20 cm/sec, and “borderline” (BL-ED) with PSV between 25 and 21 cm/sec or with PSV between 35 and 25 with concomitant EDV >0 cm/sec [The IIEF questionnaire  is a val0 cm/sec , 26. TheSerum concentrations of hs-TnT  and of hs-TnI  were measured according to the manufacturer's recommendations. The 99th percentile value for our normal reference population was 18.3 pg/mL for hs-TnT and 37 pg/mL for hs-TnI measured with a CV <10%.All patients were referred to a cardiologist for evaluation of ischemic heart disease (IHD). The evaluation consisted of a comprehensive clinical history, physical examination, and an electrocardiogram while resting and during treadmill exercise. The diagnosis of ischemic heart disease relied on the detection of a 1 mm or more horizontal or downsloping ST-segment depression, frequent ventricular premature beats, or typical chest pain during the treadmill exercise test. In addition, the Duke Treadmill Score (DTS) as developed by Mark et al.  was calcP value < 0.05 was considered significant.All the results are expressed as the mean ± standard deviation. The data were not normally distributed and were normalized by using the log or log-log transformation. Undetectable values of hs-TnT and hs-TnI concentrations were considered equal to 0 for statistical purpose. In this regard, undetectable concentrations of hs-TnT were present in 49 out of 120 patients studied (40.8%) and undetectable concentrations of hs-TnI in 78 of the 120 studied patients (65.0%). The statistical analysis was carried out by using the ANOVA test taking into account the three groups of patients. The association between IIEF-5 and laboratory parameters was examined by Pearson's correlation coefficient. The estimate beta-power was equal to 0.004. P > 0.05). All the men had DTS > +5 without significant difference between the three groups (P > 0.05).Of the 120 men included in the study, 40 patients (33.3%) were classified as having A-ED, 48 patients (40%) as having NA-ED, and, finally, 32 patients (26.6%) as having BL-ED. In A-ED (mean age 52 ± 8.5 years), NA-ED (mean age 58.1 ± 7.3 years), and BL-ED (mean age 50.0 ± 10.5 years) the mean of IIEF values was 12.8 ± 2.4, 10.5 ± 2.6, and 11.6 ± 1.8, respectively. There were no significant differences between the three groups (P > 0.05) of serum concentration of both hs-TnT and hs-TnI was found among the three groups of ED patients. The hs-CRP values were higher in A-ED men compared with NA-ED (P = 0.048) but not compared with BL-ED (P = 0.136), while no significant difference was found between BL-ED and NA-ED (P = 0.964). Spearman's correlation analysis demonstrated a negative correlation between serum hs-CRP and IIEF-5 that reached the significance in A-ED  but without reaching the significance in NA-ED and BL-ED [β-estradiol, prolactin, and testosterone)  .A consensus exists to consider ED an independent predictor of CVD –36, inclThe aim of the study was to assess the presence of cardiovascular involvement with the measurement of both hs-TnT and hs-TnI levels in ED patients in order to begin the appropriate treatment quickly following the first symptoms to prevent significant cardiovascular events.The populations studied were homogeneous without clinical evidence of atherosclerotic diseases; all men had normal ECG without ST-segment depression in exercise stress test and the DTS > +5.Our results, using the measurement of hs-TnT or hs-TnI, at least in our experimental conditions, exclude the presence of cardiovascular injury in all patients with ED for less than one year and also in those of arteriogenic etiology. The fact that no differences were found among the three groups excludes the possibility of other statistical analyses such as the exploration of an optimal cut-point to differentiate the three groups.In our patients we found that the serum concentrations of hs-CRP are higher in A-ED and the levels are associated with penile arterial disease severity assessed with IIEF-5. These data agree with those previously reported , 46–48. The number of patients included in this study seems to be low, but it must be considered that the population studied was carefully selected. In addition, the patients studied had no clinical evidence of atherosclerotic diseases and were free of the common risk factors associated with generalized penile arterial insufficiency such as hypertension, hyperlipidemia, cigarette smoking, diabetes mellitus, and pelvic irradiation. This is the first study to compare the value of troponin as a marker of cardiovascular disease before clinical symptoms in ED patients. Finally, we do not know whether troponin (T or I) may add information by adding the determination of other biomarkers such as NT-proBNP, as recently suggested , 50, or In conclusion, this study shows that the measurement of hs-TnT or hs-TnI levels allows us to exclude the presence of cardiovascular disease in patients with ED. The reason is probably the short history of ED, which was of less than one year. Longitudinal study is currently in progress to follow the level of hs-Tn (I and T) in this selected group of men, particularly in A-ED, in order to recognize when the heart begins to be affected by the process of atherosclerosis, before clinical symptoms. At present, we know the basal levels of both hs-Tn types in all our patients, and knowing the reference change values (RCV) –54, we c


Identifying latent tuberculosis infection (LTBI) in people migrating from TB endemic regions to low incidence countries is an important control measure. However, no prospective longitudinal comparisons between diagnostic tests used in such migrant populations are available.To compare commercial interferon (IFN)-gamma release assays (IGRAs) and the tuberculin skin test (TST) for diagnosing LTBI in a migrant population, and the influence of antecedent TST and LTBI treatment on IGRA performance.This cohort study, performed from February to September 2012, assessed longitudinal IGRA and TST responses in Nepalese military recruits recently arrived in the UK. Concomitant T-SPOT.TB, QFT-GIT and TST were performed on day 0, with IGRAs repeated 7 and 200 days later, following treatment for LTBI if necessary.166 Nepalese recruits were prospectively assessed. At entry, 21 individuals were positive by T-SPOT.TB and 8 individuals by QFT-GIT. There was substantial agreement between TST and T-SPOT.TB positives at baseline , but only moderate concordance between positive IGRAs . When reassessed 7 days following TST, numbers of IGRA-positive individuals changed from 8 to 23 for QFT-GIT (p = 0.0074) and from 21 to 23 for T-SPOT.TB (p = 0.87). This resulted in an increase in IGRA concordance to substantial . Thus, in total on day 0 and day 7 after testing, 29 out of 166 participants (17.5%) provided a positive IGRA and of these 13 were TST negative. Two hundred days after the study commenced and three months after treatment for LTBI was completed by those who were given chemoprophylaxis, 23 and 21 participants were positive by T-SPOT.TB or QFT-GIT respectively. When individual responses were examined longitudinally within this population 35% of the day 7 QFT-GIT-positive, and 19% T-SPOT.TB-positive individuals, were negative by IGRA. When the change in the levels of secreted IFN-γ was examined after chemoprophylaxis the median levels were found to have fallen dramatically by 77.3% from a pre-treatment median concentration of IFN-γ 2.73 IU/ml to a post-treatment median concentration IFN-γ 0.62 (p = 0.0002).This study suggests differences in the capacity of commercially available IGRAs to identify LTBI in the absence of antecedent TST and that IGRAs, in the time periods examined, may not be the optimal tests to determine the success of chemoprophylaxis for LTBI. The effective control and elimination of tuberculosis (TB) in low-incidence countries requires the prompt identification of individuals with active and latent TB infection (LTBI). This latter group has the potential to reactivate their infection, progress to disease and transmit the organism to others. In the United Kingdom (UK), TB notifications have increased to a plateau of 9,000 new cases per year of which >70% now occur in foreign-born individuals, in whom the incidence rate of TB is >20 times higher than among those born in the UK Mycobacterium tuberculosis (M.tb)-specific early secretory antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) and TB7.7 antigens has been employed in IFN-γ release assays (IGRAs). IGRAs have some advantages over TST including the speed in which results are obtained, theoretical independence from prior Bacillus Calmette-Guérin (BCG) vaccine exposure, and their potential for intra-patient repeat use Nonetheless, the diagnosis of LTBI remains challenging. Until recently, the tuberculin skin test (TST) was the diagnostic method of choice for LTBI. Although cheap and easy to perform, TST has limited sensitivity and specificity and is not suitable for repeat testing There are multiple strategies for diagnosing LTBI. Some countries recommend a single-step IGRA-based LTBI screening strategy, whilst others advocate a primary TST and subsequent IGRA in TST-positive individuals, which is the current UK recommendation M.tb-specific antigens may also provide some correlative measure of bacterial burden and so help determine the effectiveness of anti-tuberculosis treatment Once LTBI has been diagnosed and treated, clear correlates that identify whether treatment has been successful, or not, are desirable yet sadly lacking For almost two centuries the British Army has recruited young Nepalese males into the Gurkha regiment. Nepal is a region of high TB endemicity with an incidence of 163 cases per 100,000 population per year and before leaving Nepal for the UK, recruits are examined to exclude active TB disease. In the UK they are further assessed for LTBI. This provides a unique opportunity to examine IGRA responses longitudinally, with and without chemoprophylaxis, in a well-defined cohort of individuals, whose environmental experiences are controlled in a manner not normally possible in civilian populations. Additionally, all individuals can be sampled in parallel on the same day. This synchronous processing enables direct comparisons to be made between IGRAs and TST in the same patient cohort and whether chemoprophylaxis influences subsequent IGRAs and offers an opportunity to optimally diagnose LTBI in populations of migrants leaving countries of relatively high TB incidence to a country of low TB incidence. The objectives of the current study were to examine the longitudinal IGRA responses in Gurkha recruits after recent entry into the UK and the effect of TST and chemoprophylaxis on the results obtained.Ethical approval was granted by the Ministry of Defence Research Ethics Committee (MODREC 237/PPE/11). This was a prospective, longitudinal cohort study among Nepalese military recruits who had left Nepal and recently entered the UK. Inclusion criteria included age at enrolment of ≥18 years and exclusion of active TB disease, and in accordance with current military and UK migrant screening policy, HIV status was not assessed. The estimated prevalence of HIV infection among adults aged 15–49 years in Nepal is 0.3% Two commercially available assays were used - The QuantiFERON-TB Gold In-Tube  whole blood enzyme-linked immunosorbent assay (ELISA), and the T-SPOT.TB assay  enzyme-linked immunospot (ELISPOT) assay using peripheral blood mononuclear cells (PBMCs). Peripheral venous blood samples were provided at the first visit (day 0), and on days 7 and 200. Samples were transported to Oxford Immunotec's diagnostic laboratory for the T-SPOT.TB assay within 24 hours and the Health Protection Agency regional mycobacterial reference laboratory  for the QFT-GIT assay within 8 hours of collection.Standard criteria for positive responses were used for both tests as recommended by the manufacturers: for QFT-GIT, IFN-γ concentrations ≥0.35 IU/ml and ≥25% of the negative control value, after correcting for negative control values; for T-SPOT.TB, ≥6 SFCs/250,000 PBMCs to ESAT-6 or CFP-10, adjusted for the negative control, except when the negative control was ≥6 SFCs, whereupon SFCs of ≥ twice the negative control were required. Between serial IGRAs, conversion to positivity was defined as an initial IFN-γ concentration of <0.35 IU/ml or <6 SFCs which increased to ≥0.35 IU/ml or ≥6 SFCs, respectively. The opposite definition was used for reversion to negativity. ‘Borderline’ or ‘uncertainty’ zones have previously been defined as 0.2–0.7 IU/ml for QFT-GIT or 4–8 SFCs for T-SPOT.TB. Conversion thresholds of an increase of <0.2 to >0.7 IU/ml for QFT-GIT and from <6 to >9 SFCs for T-SPOT.TB, were also applied in our analyses On day 0, following blood sampling for the IGRAs, a TST was performed by the Mantoux method. Two tuberculin units of strain RT23 purified protein derivative  was applied intradermally and transverse induration measured 72 hours later. Positivity was defined as an induration of ≥6 mm in BCG-naive and ≥15 mm in BCG-vaccinated individuals in accordance with UK guidelines (stratified threshold). We also used non-stratified threshold for TST positivity, defined as induration ≥10 mm irrespective of BCG vaccination history.Individual's positive for either IGRA at day 0 and/or day 7, with normal chest radiography and no indication of active disease, were defined as having LTBI and offered LTBI treatment. Individuals who only provided a positive TST, but not a positive IGRA, were not treated. A three month regimen of daily self-administered weight-based, fixed-dose combination therapy of rifampicin and isoniazid was prescribed in accordance with current UK guidance κ≤0.20, ‘fair’ for 0.20<κ≤0.40, ‘moderate’ for 0.40<κ≤0.60, ‘substantial’ for 0.60<κ≤0.80 and ‘optimal’ for 0.80<κ≤1.00 Data analyses were performed using GraphPad Prism. The two-tailed Fisher's exact test was used to compare categorical data and the Mann-Whitney test (unpaired) or Wilcoxon signed rank test (matched-pairs) for analysis. McNemar's test was used to compare paired proportions. Concordance between TST and IGRA results was assessed using percentage agreement and Cohen's κ coefficients; strength of agreement was considered ‘poor’ for Of 176 Gurkha recruits who arrived in the UK from Nepal, 166 volunteered to participate in the study. All participants were male, aged 18–21 years and 105 had been vaccinated with BCG. No participant reported a prior history of TB, known exposure to TB or any other past medical history. All individuals enrolled at day 0 re-attended at days 3, 7 and 200 and provided a complete set of samples . In eachThe IGRA testing revealed that 21 individuals were T-SPOT.TB positive  and eight were QFT-GIT positive (4.8%). All QFT-GIT positives were also T-SPOT.TB positive. Thus, 61.9% T-SPOT.TB positive participants were QFT-GIT negative, meaning the tests showed moderate agreement  .2 = 0.88 and 0.95, respectively; TST were performed at day 0 and read at day 3, and the results compared to the day 0 IGRAs. There was substantial agreement between TST and T-SPOT.TB and fair agreement between TST and QFT-GIT . Twenty-To examine if the TST influenced the results of a subsequent IGRA, repeat IGRAs were performed 7 days after TST administration. Four (19.0%) of the 21 T-SPOT.TB positive individuals at day 0 were no longer positive on repeat IGRA, two of whom had been ‘borderline positive’ on day 0. Six (4.2%) of the 142 IGRA-negative individuals on day 0 became positive by T-SPOT.TB, including one ‘borderline positive’ result, yielding a total of 23 positives at day 7 (13.9%). One of six converters was TST positive.None of the eight day 0 positive QFT-GIT individuals reverted to negativity. However 15 (9.5%) previously negative individuals converted to positivity, nearly tripling the numbers of positive individuals to 23 (13.9%) on day 7 (p = 0.0074). Of the 23 QFT-GIT-positive individuals, nine were also TST-positive (60.0% concordance). Three of six T-SPOT.TB converters also provided positive QFT-GIT tests, thus increasing the agreement between the IGRA tests and TST to substantial  . ConcordSince antecedent TST could alter IGRA positivity we also assessed whether the strength of the IGRA response was also affected. Changes in positive results were observed for both IGRA tests and were often, though not always, associated with increased numbers of results falling into the zones of uncertainty . The eigAmong the six individuals converting to T-SPOT.TB positivity at day 7, numbers of SFCs increased for both ESAT-6 and CFP-10, with medians rising by up to 3-fold . Among the 17 T-SPOT.TB-positives at both day 0 and 7, median SFCs for each antigen over the two sampling points remained relatively stable . However, among the eight individuals positive by both IGRAs at days 0 and 7 there was augmentation in SFC numbers for both antigens, but particularly to ESAT-6 . There was no significant change in SFC numbers in the 134 persistently negative individuals.A significant proportion of the dynamic IGRA responses (conversions and reversions) following TST occurred around the zones of uncertainty for both assays . HoweverAfter excluding the presence of active disease, each of the 29 individuals considered positive for LTBI, by either one or both IGRAs at either day 0 or day 7, underwent and completed treatment without complication. Three months after completion the whole cohort provided a third sample. Five of the 27 individuals who provided a positive T-SPOT.TB IGRA at day 0 or day 7 or both times reverted to negativity after they completed treatment . In those positive by T-SPOT.TB, numbers of ESAT-6 or CFP-10 SFC were not markedly different . Of the Of the 23 individuals positive by QFT-GIT at either day 0 or day 7 or both times, eight reverted to negativity after they had completed their treatment. Median IFN-γ levels decreased by 77.3% following treatment . Of the eight individuals initially positive at day 0, two became negative , while of the 15 participants who became positive on day 7 after the TST, 40% reverted  . Six of This study evaluated the performance of currently available commercial IGRAs for the diagnosis of LTBI in a migrant population, and how antecedent TST and LTBI treatment influence these. The key strengths of this study are the cohort, the similar population age and ethnicity, the assured adherence to treatment, the synchronous manner in which samples were collected and processed and the setting that controlled for TB exposure.At first presentation nearly three times more individual's had positive TST and T-SPOT.TB results than QFT-GIT results. For all tests there was a greater concordance between an IGRA test and TST in BCG-naive than BCG-vaccinated individuals , as prevA lack of a ‘gold standard’ test introduces difficulties in evaluating the performance and diagnostic accuracy of assays for detecting LTBI Multiple studies suggest that the T-SPOT.TB assay has a higher sensitivity but somewhat lower specificity than QFT-GIT Administration of PPD resulted in a marked increase in QFT-GIT positive individuals, which was not seen for the T-SPOT.TB test. It is unlikely the more numerous T-SPOT.TB positives at day 0 are false positives due to the greater overall diagnostic agreement between TST and T-SPOT.TB results than between TST and QFT-GIT. Additionally, longitudinal, intra-individual T-SPOT.TB results were generally consistent and the TST resulted in QFT-GIT results more reflecting those of the T-SPOT.TB test. Technical factors are unlikely to have influenced QFT-GIT results at day 0 because samples were collected, transported and processed similarly each time. Also, controls were within expected ranges, no indeterminate QFT-GIT results were recorded and the assay was performed by a national TB reference laboratory. We interpret these results to indicate that QFT-GIT has a lower sensitivity for diagnosing LTBI and that tuberculin may enhance the response in some individuals to this test. Further carefully controlled studies are required to confirm these findings.Results from IGRAs can be dynamic, producing transient responses. Intra-subject variability can complicate their interpretation, particularly for borderline cases, and may occur for reasons including exposure to active TB cases and environmental NTM Our data suggest that by assessing IGRA responses before and after TST, five distinct groups can be identified . The firM.tb prior to the study, with the results reflecting weak responses declining with time Among those who converted to positivity during the study we believe there are two groups. The first is comprised of those negative at baseline for both IGRAs and a second group that had discordant IGRA results at baseline. Expansion of memory T cells following PPD stimulation may account for conversion among the first group. The differences in these groups may relate to the level of bacterial burden or antigen availability and therefore the level of immune stimulation. A low sensitivity assay or a weak immune response may produce initial false negative results, which is enhanced after further antigen exposure (i.e. PPD). Differences between the whole-blood ELISA and PBMC ELISPOT may also contribute to the inter-assay variation observed. The final, fifth group of positive to negative reversions only occurred in discordant individuals. One possible explanation is that this may reflect transient exposure to Following completion of treatment, 35% of QFT-GIT-positive individuals reverted to negativity. Median IFN-γ levels decreased significantly in those treated but the change was most profound in those who were positive only after antecedent TST and least marked in those who were positive on day 0. In contrast, responses in the T-SPOT.TB assay were much less affected by chemoprophylaxis. Previous studies have reported variable results for the T cell response to ESAT-6 and CFP-10 in adults following treatment for LTBI for reasons that may include the differing methodologies used and populations examined between studies There are a number of limitations to this study. First is the lack of a true ‘gold standard’ for diagnosing LTBI which is an issue shared with other studies in this field. Furthermore, there was no untreated LTBI control group and also no regular, longitudinal follow-up of individual patients. Additionally, although unlikely to have any persisting effect, antecedent TST administration may have confounded the response of an individual to treatment.In summary, this study has examined the response in two commercially available IGRAs for the diagnosis of LTBI in a migrant population of Gurkha recruits. The study finds that in the absence of a TST then more positive responses could be identified by the T-SPOT.TB test than by QFT-GIT. Nevertheless, after a recent TST the responsiveness to both tests was more similar, mainly due to an increase in the numbers of positive QFT-GIT responses. Follow-up IGRA testing for LTBI after treatment in this population supports the current concept that monitoring of IGRA responses in adults following completion of chemoprophylaxis is of speculative value only.In light of the findings presented in this study the UK Ministry of Defence has altered its policy for LTBI screening in Gurkha recruits to the use of a single T-SPOT.TB IGRA


N-methyl-D-aspartate receptors (NMDARs) are major glutamate receptors in the striatum that receive glutamatergic projections from the frontal cortex. The membrane insertion of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) depends on NMDAR activation and is required in learning and memory processes. In the present study, we measured set-shifting and reversal learning performance in operant chambers in rats and assessed the effects of blocking NMDARs and Ca2+-permeable AMPARs in striatal subregions on behavioral flexibility. The blockade of NMDARs in the nucleus accumbens (NAc) core by AP5 impaired set-shifting ability by causing a failure to modify prior learning. The suppression of NMDAR-mediated transmission in the NAc shell induced a deficit in set-shifting by disrupting the learning and maintenance of novel strategies. During reversal learning, infusions of AP5 into the NAc shell and core impaired the ability to learn and maintain new strategies. However, behavioral flexibility was not significantly affected by blocking NMDARs in the dorsal striatum. We also found that the blockade of Ca2+-permeable AMPARs by NASPM in any subregion of the striatum did not affect strategy switching. These findings suggest that NMDAR-mediated glutamate transmission in the NAc contributes more to cognitive execution compared with the dorsal striatum.Cognitive flexibility is a critical ability for adapting to an ever-changing environment in humans and animals. Deficits in cognitive flexibility are observed in most schizophrenia patients. Previous studies reported that the medial prefrontal cortex-to-ventral striatum and orbital frontal cortex-to-dorsal striatum circuits play important roles in extra- and intra-dimensional strategy switching, respectively. However, the precise function of striatal subregions in flexible behaviors is still unclear.  Strategy switching as a cognitive ability is critical for adapting to and surviving in an ever-changing environment. Impaired cognitive flexibility is one of the core symptoms of schizophrenia  selectively impairs behavioral flexibility without influencing initial reward-response association learning -amino-5-phosphonopentanoate (AP5) or GluA2-lacking AMPAR antagonist 1-naphthylacetylspermine (NASPM) into subregions of the striatum before performing the behavioral tasks. Four subregions of the striatum are the nucleus accumbens (NAc) core, NAc shell, DMStr, and DLStr. Traditionally, the term “striatum” refers to the dorsal striatum. The NAc is referred to as the ventral striatum because it is considered to be the predominant part of the ventral striatum. The shell and core subregions of the NAc have different connectivity, with the shell more closely related to the limbic system and the core more closely related to the dorsal striatum , weighing 280–300 g upon arrival, were used in the study. The rats were individually housed in stainless-steel sliding-drawer-type cages (50 cm length × 32 cm width × 22 cm height) with a wire mesh floor and pine wood-shavings below the cages. The rats were maintained on a 12/12 h light/dark cycle (lights on at 7:00 AM), with water and food available The behavioral experiments were conducted in eight operant chambers  enclosed in sound-attenuating boxes. Each chamber was fitted with two nosepoke operandi, one of which was located on each side of a central liquid receptacle. Two yellow light-emitting-diode cue lights (20 mW) were separately situated inside the nosepoke holes. A white chamber light was located 20 cm above the right nosepoke hole. Solutions were delivered through a metal spout that was attached to a 60-ml syringe pump with tubing that delivered fluid at a rate of 34.450 ml/min. The pumps were calibrated to dispense 0.08 ml (0.139 s) of solution into a liquid receptacle for each reinforcement. A timeout period of 10 s followed each reinforcement, during which further nosepokes produced no effect.The rats were anesthetized with sodium pentobarbital  before cannula implantation surgery. After being placed in a stereotaxic frame, a midsagittal incision was made to retract the scalp and expose the cranium. Bilateral 23-gauge stainless-steel guide cannulas  were implanted into the NAc core , NAc shell , or DLStr . The coordinates were based on the Paxinos and Watson  rat braiAt the beginning of habituation, the rats were placed in the operant chambers. They were allowed to freely explore the chamber for 15 min with the nosepoke light off. Sucrose reinforcement was unavailable during this period. A 60-min session was used to train the rats to learn that the sucrose solution was available in the central liquid receptacle. The chamber was illuminated by two nosepoke lights, and liquid was delivered into the central liquid receptacle on a variable interval (VI) 40-s schedule  turned on the house light and switched on the syringe pump 1 s later. Nosepokes in the other hole (inactive hole) had no scheduled consequences. The acquisition criterion was defined as nine consecutive correct responses. A similar criterion was used in previous studies , regardless of the location of the stimulus light that was illuminated previously in one of the nosepoke holes. Similar to the initial visual-cue discrimination, in every pair of trials, the left or right stimulus light was illuminated once, and the order was random within the pair of trials. The trials were conducted identically to the initial visual-cue discrimination trials. In each trial, the nosepoke hole that the animal chose and the location of the stimulus light were recorded. The trials continued until either (1) the rat achieved criterion performance of nine consecutive correct responses or (2) 100 trials were completed. In this experiment, all of the rats achieved criterion performance within this allotted number of trials.One day after the set-shifting trials, the rats were required to learn a reversal of the response rule. The rats had to nosepoke in the hole opposite to the one they learned during the set-shifting task on the preceding day. Thus, if a rat was required to nosepoke in the left hole during the set-shifting task, then it had to nosepoke in the right hole in the reversal learning task, regardless of the position of the visual cue. The other aspects of the training were identical to the response discrimination training day. One of the visual-cue lights was illuminated prior to the start of each trial and served as a distracting stimulus.We performed an error analysis of the set-shifting and reversal-learning data as reported previously , AP5 , or their corresponding vehicle solution (0.9% NaCl solution). Saline or the antagonist was infused at a rate of 0.25 μl/100 s (NASPM) or 0.5 μl/100 s (AP5) by a microsyringe pump. The injection needles were left in place for an additional 1 min to allow for drug diffusion. Following the infusion, the rats were placed in the operant chamber. Behavioral testing commenced 5 min (NASPM) or 15 min (AP5) later. NASPM and AP5 were purchased from Sigma.After behavioral testing, the rats were sacrificed in a carbon dioxide chamber. The brains were removed, fixed in a 4% formalin solution, frozen, sliced into 50 μm coronal sections, mounted on slides, and stained with Cresyl violet. The needle placements were verified with reference to the neuroanatomical Paxinos and Watson  rat braipost hoc tests. Subsequent targeted analyses that compared the numbers of each type of error were conducted using two-way ANOVAs. The effects of AP5 on simple learning were analyzed using two-tailed t-tests per NAc subregion. The criterion for statistical significance was set to p < 0.05.The data were analyzed using GraphPad Prism 6.0 software. The numbers of trials and errors to reach criterion were separately analyzed using two-way analysis of variance (ANOVA) for each brain region, with Treatment as the within-subjects factor and Training phase as the between-subjects factor. Significant main effects in the ANOVA were followed by Bonferroni multiple-comparison F = 9.441, p < 0.01; Figure F = 14.54, p < 0.001; Figure p < 0.05) and made more errors (p < 0.01) compared with saline infusion during reversal learning. AP5 infusion significantly increased the number of errors to reach criterion (p < 0.05) but not the number of trials to reach criterion (p > 0.05) during the set-shifting task. Subsequent analysis of the types of errors during the set-shifting task made by the rats that received AP5 revealed a significant effect of Treatment  = 4.392, p < 0.05; Figure p < 0.05) but not regressive errors or never-reinforced errors (p > 0.05). In the reversal learning task, the analysis of the types of errors also showed a significant effect of Treatment  = 5.624, p < 0.05; Figure p < 0.05) and not perseverative errors (p > 0.05). Two subtypes of regressive errors were further analyzed by two-way ANOVA. The results showed a significance increase in the number of regressive errors toward the visual cue (p < 0.05) and a trend toward an increase in the number of regressive errors away from the visual cue.Fifty-three rats were trained to obtain 20% sucrose solution by nosepoking the hole associated with the visual cue. Twenty-four hours later, 15 rats were infused with AP5 into the NAc core before the set-shifting task, and 36 rats were infused with the corresponding saline vehicle. Twenty-four hours after the set-shifting task, the rats that received saline treatment during the set-shifting task were divided into an AP5 infusion group (19 rats) and saline infusion group (17 rats) for the reversal learning task. The two-way ANOVA of these data revealed a significant effect of Treatment on both the number of trials to reach criterion  = 9.426, p < 0.01; Figure F = 9.96, p < 0.01; Figure p < 0.05). Subsequent targeted analysis of the types of errors made during the set-shifting task after AP5 infusion revealed a significant effect of Treatment  = 2.494, p < 0.05; Figure p < 0.05), without affecting perseverative or never-reinforced errors (p > 0.05). In the reversal learning task, the analysis of the types of errors also revealed a significant effect of Treatment  = 5.662, p < 0.05; Figure p < 0.001). Two subtypes of regressive errors were analyzed by two-way ANOVA. The results showed a significance increase in the number of regressive errors toward the visual cue (p < 0.05) and a trend toward an increase in the number of regressive errors away from the visual cue.The effects of AP5 infusion in the NAc shell on behavioral flexibility are shown in Figure t-tests for each kind of learning task based on each brain region. Figure t < 1.1, no significant differences).To assess whether the blockade of NMDARs in the NAc affects the initial learning associated with visual-cue and spatial discrimination, we infused AP5 into the NAc core and shell, respectively, before initial training. The data were analyzed using separate F = 0.3002, p > 0.05; Figure F = 2.7956, p > 0.05; Figure F = 0.0822, p > 0.05; Figure F = 0.1698, p > 0.05; Figure The effects of infusions of saline and AP5 into the DMStr and DLStr on set-shifting and reversal learning are presented in Figure F = 0.6595, p > 0.05; Figure F = 0.5259, p > 0.05; Figure F = 0.0089, p < 0.01; Figure p < 0.05), without affecting regressive or never-reinforced errors.Similarly, no difference was found between the AP5-treated and control groups in the number of trials  = 0.2662, p > 0.05, Figure F = 2.64, p > 0.05, Figure F = 0.0064, p > 0.05, Figure F = 0.0005, p > 0.05, Figure F < 1.9, no significant differences). A subsequent analysis of the types of errors made during the set-shifting and reversal learning tasks revealed no difference between the NASPM- and saline-infused groups. These data indicate that Ca2+-permeable AMPARs in the striatum do not play a crucial role in behavioral flexibility. The dose of NASPM used in the present study has been shown previously to affect reward-associated behavior . The NASPM solution was infused into the NAc core, NAc shell, DMStr, or DLStr 5 min before testing in separate experiments. Twenty-four hours after the set-shifting task, half of the rats, which were infused with saline during the set-shifting task, were infused with NASPM solution before the reversal learning task. The other half of the rats were administered saline as a control. The results are presented in Figures The present study revealed that the blockade of NMDARs in the NAc core impaired set-shifting performance by increasing perseverative errors. The suppression of NMDAR-mediated neurotransmission in the NAc shell induced a deficit in set-shifting by enhancing regressive errors. These findings indicate that the activation of NMDARs in the NAc core alters prior learning or inhibits responses that are no longer appropriate, whereas NMDARs in the NAc shell play an important role in learning and maintaining novel strategies. Reversal learning reflects intradimensional strategy switching and was tested 24 h after the set-shifting task. AP5 infusion in the NAc shell and core significantly increased the total number of trials to reach criterion and the number of regressive errors, indicating that AP5-treated rats exhibited dysfunction in novel strategy learning or maintenance. In contrast, no significant alteration was induced by AP5 infusion into the DMStr or DLStr in the set-shifting or reversal learning task. We also tested the selective GluA2-lacking AMPAR antagonist NASPM to assess the effects of blocking GluA2-lacking AMPARs in different striatal subregions on behavioral flexibility. No effect was found during the set-shifting or reversal learning task.Schizophrenia patients exhibit impaired cognitive flexibility measured by the Wisconsin Card Sorting Test  and synaptic changes can be modulated by endocytosis of GluA2-containing AMPARs (Lim et al., In summary, pharmacological tools associated with glutamate transmission may provide a potential clinical treatment for schizophrenia. In addition to the frontal cortex, the ventral striatum may be another important brain region for adjusting behavioral flexibility. These findings suggest that NMDAR-mediated glutamate transmission in the ventral striatum makes more of a contribution to cognitive execution than the dorsal striatum.Xuekun Ding, Yanhua Qiao, and Chenji Piao performed experiments, analyzed data and wrote the manuscript. Xigeng Zheng and Zhengkui Liu managed the literature searches and analyses. Jing Liang designed the study, wrote the protocol and finalized the manuscript. All authors contributed to and have approved the final manuscript and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our previous study used freeze-drying and biotin–avidin binding methods and obtained nontargeted nanobubbles (N-NBs) and ovarian cancer-targeting nanobubbles . Our study also identified the physical and chemical properties of these two contrast agents, and validated the targeting ability and underlying mechanisms of LHRH-NBs in vitro. The present study investigated the imaging of N-NBs and LHRH-NBs in nude mice and their binding with tissues.The nude mice models of xenografts were divided into three groups, N-NB, LHRH-NB, and SonoVue. These contrast agents were injected via the caudal vein to observe the imaging of ovarian cancer. Fluorescence microscope was used to observe the penetration of N-NBs and LHRH-NBs through the vascular endothelial gaps. Immunofluorescence was used to observe the penetration of N-NBs and LHRH-NBs through vascular endothelial gaps and binding to the tumor cells.P < 0.001). Both N-NBs and LHRH-NBs penetrated through the vascular endothelial gaps. After penetrating through the vascular endothelial gapes, LHRH-NBs could target and bind to the tumor cells.The imaging intensity and duration were not significantly different between N-NBs and LHRH-NBs. The imaging intensity in the N-NB and LHRH-NB groups was not significantly different compared with the SonoVue group; however, the imaging duration in the N-NB and LHRH-NB groups was significantly longer than in the SonoVue group (N-NBs and LHRH-NBs are of good imaging effectiveness and relatively long imaging duration. LHRH-NB is a potent contrast agent for imaging ovarian cancer, while achieving targeted delivery of drugs to the site of ovarian cancer. Ovarian cancer when compared to other gynecology-associated malignancies has the highest mortality rate . As the As a real-time, noninvasive, and an accurate imaging method, ultrasonic contrast technology has wide applications in clinical practice . With thIn light of these findings, in our previous studies , we chos3F8) was purchased from the Tianjin Research Institute of Physical and Chemical Engineering of Nuclear Industry . OVCAR-3 cells were obtained from Huiying Biological Technology Co. LTD. . Avidin and McCoys 5A culture medium were obtained from Boster Biological Technology Co. LTD. . Blocking serum, 4,6-diamidino-2-phenylindole (DAPI), and cell membrane red fluorescent probe (DiI) were from Beyotime Biological Technology Co. LTD. . SonoVue was obtained from Bracco Imaging B.V. Company . BALB/c nude mice were from the Experimental Animal Center of Chongqing Medical University, and the protocols in this study were approved by the Ethics Committee of Chongqing Medical University. The DFY ultrasound image quantitative analysis of diagnostic equipment was provided by Institute of Ultrasound Imaging, Chongqing Medical University. Philips iU 22 was from Philips . CKX41 inverted fluorescence microscope was from Olympus Company .Dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylethanolamine (DSPE), and biotinylated dipalmitoyl phosphatidylethanolamine (DSPE-PEG2000-Biotin) were obtained from Avanti Company . Biotinylated LHRH antibody and FITC-labeled goat anti-rabbit immunoglobulin (Ig) G were from Beijing BiossBiological Technology Co. LTD. . Perfluorinated propane  were cultured in McCoys 5A medium containing 10 % heat-inactivated fetal calf serum at 37 °C and incubated in 5 %COWe have successfully prepared nanoscale liposome microbubbles, namely N-NBs and LHRH-NBs, in our previous studies  via free7/ml. Then, 0.2 ml of the suspension was subcutaneously injected into the right hip of the mice. After the tumor reached a size of 1.0 cm, the following experiments were performed.Thirty female BALB/c nude mice aged 4–6 weeks were obtained. OVCAR-3 cells in the logarithmic growth phase were collected to obtain cell suspension with a density of 1 × 106) were resuspended in 200 μl phosphate-buffered saline (PBS), and then, the suspension was injected via the caudal vein. The ultrasound probe was placed at the largest transverse section of the tumor, the imaging of the tumor was dynamically observed, and the images were preserved. Equal volume of the other two contrast agents, namely LHRH-NBs and SonoVue, were also injected via the caudal vein, and the data were recorded under the nontargeting contrast agent model. DFY ultrasound image quantitative analysis of diagnostic equipment was used to analyze the images.The mice were anesthetized with 10 % chloral hydrate. The mice were then fixed on the operating table, and body temperature was maintained using a heater. N-NBs  was added. The slices were incubated for 30 min in dark, and the fluorescence was observed. Same procedures were applied for N-NBs to obtain the slices and observe the fluorescence. For the blank control, the tumor tissues were harvested without injection of contrast agent, and then, the rapid-frozen slices, 5 μm thick, were prepared. These slices were also washed with PBS thrice for 3 min each time and blocked with blocking serum for 30 min at 37 °C, and then, primary LHRH antibody (1:100) was added and incubated at 4 °C overnight. The slices were then placed at 37 °C for 30 min and washed with PBS thrice for 5 min each time. FITC-labeled goat anti-rabbit IgG (1:100) was then added and incubated for 30 min in dark, and fluorescence microscope was used to observe the fluorescence. The absorption wavelength of FITC was 490 nm, and the emission wavelength was 520 nm.P < 0.05 was considered statistically significant.SPSS 19.0 software was used for statistical analysis. Quantitative data are described as mean ± SD. Analysis of variance was used for comparisons between different groups. In our previous studies , the twoOur previous studies  demonstrP > 0.05). However, the imaging duration was longer in N-NBs and LHRH-NBs than SonoVue (Table P < 0.001). Figure No tumor-bearing nude mice died during the experiment. The dynamic changes in the three contrast agents, namely N-NBs, LHRH-NBs, and SonoVue, with time  are shown in Figs. Figure The results of immunofluorescence examinations in the LHRH-NB group are shown in Fig. With the development of ultrasonic contrast technology, the molecular imaging technology has shown great advantages in the early diagnosis of diseases. The evaluation of the imaging effectiveness of a contrast agent is mainly dependent on the following aspects: imaging intensity, imaging duration, and background interference. Micron-sized contrast agents are with relatively large particle size and gas contents, and the imaging intensity should be better than nanoscale contrast agents theoretically. However, the findings of the present study showed that the imaging effectiveness was not significantly different between the two nanoscale contrast agents (N-NBs and LHRH-NBs) and the micron-sized contrast agent SonoVue. The reasons could be as follows: (1) Nanoscale contrast agents visualize not only the vessel pool but also the tissues, and the latter could compensate the weakness of the nanoscale contrast agents in the intravascular imaging, and (2) when circulating in the body of the nude mice, the body temperature of the mice was higher than room temperature, the size of the nanoscale contrast agents could increase in a relatively high temperature, and some of them could even increase to almost the size of micron-sized contrast agents. Deshpande et al.  have shoBoth N-NBs and LHRH-NBs are nanoscale contrast agents, but which one has better imaging effectiveness? We hypothesized that after reaching the target regions, LHRH-NBs, the contrast agent with extremely high penetration ability, could bind to the LHRH receptor expressed on the surface of the ovarian cancer cells through the LHRH antibody on the surface of the LHRH-NBs, and actively adhere to the tumor cells for a long time and aggregate in the tissue cells. Our previous studies have demonstrated the in vitro targeting ability of LHRH-NBs, which was confirmed by results from the fluorescence microscope and immunofluorescence examinations in the present study. The aggregation imaging evidently increased the signals in the targeted tumor regions and also maintained low background noise. Therefore, compared with N-NBs, LHRH-NBs theoretically have higher imaging intensity and longer imaging duration, and thus be a promising contrast agent in molecular imaging of ovarian cancer. However, the findings of the present study showed that the imaging effectiveness was not significantly different between the N-NB and LHRH-NB groups, and the reasons could be as follows: (1) both N-NBs and LHRH-NBs are identically affected by the hemodynamics in the blood vessels, and thus, the effects on the intravascular imaging are negligible; (2) the binding of LHRH-NBs to the tumor cells requires some time, while some bubbles could rupture even before binding to the tissues, thus the binding rate of LHRH-NBs to the tissue cells could be limited; and (3) the mobility of the tissue liquid is relatively low, thus N-NBs could remain in the tissue spaces even without actively binding to the cells and thus imaging the tissues. These factors finally caused the numbers of the two nanobubbles in the imaging of the tissues to be not significantly different; thus, compared with N-NB group, the imaging intensity and duration in the LHRH-NB group was not significantly different.Although LHRH-NBs manifested no outstanding imaging advantages compared with N-NBs, fluorescence microscopy and immunofluorescence examinations in the present study showed that LHRH-NBs could penetrate through the endothelial gaps, followed by targeting and binding to ovarian cancer cells. Therefore, it is possible to use these nanoscale microbubbles as a vehicle to load the drugs and thus perform targeted therapy for ovarian cancer. We hypothesized that after penetrating through the newly developed tumor blood vessels with relatively loose endothelial spaces , the LHR